Labelled nucleotides

ABSTRACT

The invention provides a nucleotide or nucleoside having a base attached to a detectable label via a cleavable linker, characterised in that the cleavable linker contains a moiety selected from the group comprising: Formula (I) (wherein X is selected from the group comprising O, S, NH and NQ wherein Q is a C 1-10  substituted or unsubstituted alkyl group, Y is selected from the group comprising O, S, NH and N(allyl), T is hydrogen or a C 1-10  substituted or unsubstituted alkyl group and * indicates where the moiety is connected to the remainder of the nucleotide or nucleoside).

FIELD OF THE INVENTION

This invention relates to labelled nucleotides. In particular, thisinvention discloses nucleotides having a removable detectable label andtheir use in polynucleotide sequencing methods.

BACKGROUND

Advances in the study of molecules have been led, in part, byimprovement in technologies used to characterise the molecules or theirbiological reactions. In particular, the study of the nucleic acids DNAand RNA has benefited from developing technologies used for sequenceanalysis and the study of hybridisation events.

An example of the technologies that have improved the study of nucleicacids, is the development of fabricated arrays of immobilised nucleicacids. These arrays consist typically of a high-density matrix ofpolynucleotides immobilised onto a solid support material. See, e.g.,Fodor et al., Trends Biotech. 12:19-26, 1994, which describes ways ofassembling the nucleic acids using a chemically sensitized glass surfaceprotected by a mask, but exposed at defined areas to allow attachment ofsuitably modified nucleotide phosphoramidites. Fabricated arrays canalso be manufactured by the technique of “spotting” knownpolynucleotides onto a solid support at predetermined positions (e.g.,Stimpson et al., Proc. Natl. Acad. Sci. USA 92:6379-6383, 1995).

A further development in array technology is the attachment of thepolynucleotides to the solid support material to form single moleculearrays. Arrays of this type are disclosed in International Patent App.WO 00/06770. The advantage of these arrays is that reactions can bemonitored at the single molecule level and information on large numbersof single molecules can be collated from a single reaction.

For DNA arrays to be useful, the sequences of the molecules must bedetermined. U.S. Pat. No. 5,302,509 discloses a method to sequencepolynucleotides immobilised on a solid support. The method relies on theincorporation of 3′-blocked bases A, G, C and T, each of which has adistinct fluorescent label, into the immobilised polynucleotide, in thepresence of DNA polymerase. The polymerase incorporates a basecomplementary to the target polynucleotide, but is prevented fromfurther addition by the 3′-blocking group. The label of the incorporatedbase can then be determined and the blocking group removed by chemicalcleavage to allow further polymerisation to occur.

Welch et al. (Chem. Eur. J. 5(3):951-960, 1999) describes the synthesisof nucleotide triphosphates modified with a 3′-O-blocking group that isphotolabile and fluorescent. The modified nucleotides are intended foruse in DNA sequencing experiments. However, these nucleotides proved tobe difficult to incorporate onto an existing polynucleotide, due to aninability to fit into the polymerase enzyme active site.

Zhu et al. (Cytometry 28:206-211, 1997) also discloses the use offluorescent labels attached to a nucleotide via the base group. Thelabelled nucleotides are intended for use in fluorescence in situhybridisation (FISH) experiments, where a series of incorporatedlabelled nucleotides is required to produce a fluorescent “bar code”.

WO99/57321 describes the use of nucleotides comprising fluorophoreslinked to the nucleotide by chemically or photochemically cleavablelinker moieties.

WO00/53812 and EP-A2-1 291 354 disclose nucleotide compounds of generalstructure Fluorophore-S—S-Linker-Nucleotide and their use in nucleicacid assay methods. WO00/53812 also makes reference to periodatecleavage of a cis-glycol linkage between nucleotide and fluorophore.

WO 01/92284 discloses the use of enzyme-cleavable groups linkingblocking and reporting moieties to nucleotides. It is preferred thatthese enzyme-cleavable groups are the same, i.e. that both the blockingand reporter moieties are attached to the nucleotide by a chaincomprising a group cleavable by a common enzyme. Cleavable groupsdescribed in WO 01/92284 are esters and amides, cleavable by esterasesand amidases respectively.

WO02/29003 describes nucleotides analogues that contain a label linkedthrough cleavable linkers to the nucleotide base, or an analogue of thebase. Photocleavable linkers comprising 2-nitrobenzyl moieties aredescribed.

SUMMARY OF THE INVENTION

The present inventors have surprisingly found that certain linkers whichconnect the bases of nucleotides to detachable labels, e.g.fluorophores, may be cleaved using water-soluble phosphines orwater-soluble transition metal catalysts formed from a transition metaland at least partially water-soluble ligands. In aqueous solution thelatter form at least partially water-soluble transition metal complexes.

Labelled nucleotides comprising such linkers an methods for using themare clearly advantageous in the context of techniques carried out inaqueous media such as sequencing reactions, polynucleotide synthesis,nucleic acid amplification, nucleic acid hybridization assays, singlenucleotide polymorphism studies, and other techniques using enzymes suchas polymerases, reverse transcriptases, terminal transferases, or otherDNA modifying enzymes. The invention is especially useful in techniquesthat use labelled dNTPs, such as nick translation, random primerlabeling, end-labeling (e.g., with terminaldeoxynucleotidyltransferase), reverse transcription, or nucleic acidamplification.

According to a first aspect of the invention, there is provided anucleotide or nucleoside having a base attached to a detectable labelvia a cleavable linker, characterised in that the cleavable linkercontains a moiety selected from the group comprising:

(wherein X is selected from the group comprising O, S, NH and NQ whereinQ is a C₁₋₁₀ substituted or unsubstituted alkyl group, Y is selectedfrom the group comprising O, S, NH and N(allyl), T is hydrogen or aC₁₋₁₀ substituted or unsubstituted alkyl group and * indicates where themoiety is connected to the remainder of the nucleotide or nucleoside).

According to a second aspect of the invention, there is provided amethod of cleaving a linker that contains a moiety selected from thegroups comprising:

(wherein X is selected from the group comprising O, S, NH and NQ whereinQ is a C₁₋₁₀ substituted or unsubstituted alkyl group, Y is selectedfrom the group comprising O, S, NH and N(allyl), T is hydrogen or aC₁₋₁₀ substituted or unsubstituted alkyl group and * indicates where themoiety is connected to the remainder of a nucleotide or nucleoside),said linker being present in the nucleotide or nucleoside and connectingthe base thereof to a detectable label, said method comprisingcontacting the nucleotide or nucleoside with a water-solublephosphine-based transition metal catalyst.

According to a third aspect of the invention, there is provided a methodof cleaving a linker that contains a moiety selected from the groupscomprising:

(wherein X is selected from the group comprising O, S, NH and NQ whereinQ is a C₁₋₁₀ substituted or unsubstituted alkyl group, T is hydrogen ora C₁₋₁₀ substituted or unsubstituted alkyl group and * indicates wherethe moiety is connected to the remainder of a nucleotide or nucleoside),said linker being present in the nucleotide or nucleoside and connectingthe base thereof to a detectable label, said method comprisingcontacting the nucleotide or nucleoside with a water-soluble phosphine.

The method according to the second and third aspects of the inventionare particularly useful in sequencing reactions. Such reactionsconstitute a further aspect of the invention. Viewed from this aspect,the invention provides a method for determining an identity of anucleotide in a target single-stranded polynucleotide, comprising:

(a) providing one or more of the nucleotides A, G, C and T or U in whicheach of said nucleotides has a base that is attached to a distinctdetectable label via a linker, said linker being cleavable with awater-soluble phosphine; and a nascent polynucleotide complementary tothe target polynucleotide, one of said provided nucleotides beingsuitable for incorporation into said nascent polynucleotide;

(b) incorporating the nucleotide suitable for incorporation into saidnascent polynucleotide; and

(c) carrying out a method according to the second or third aspect of theinvention.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows exemplary nucleotide structures useful in the invention.For each structure, X can be H, phosphate, diphosphate or triphosphate.R₁ and R₂ can be the same or different, and can be selected from H, OH,or any group which can be transformed into an OH.

FIG. 2 shows some functional molecules useful in the invention, whichinclude some cleavable linkers. In these structures, R₁ and R₂ may bethe same or different, and can be H, OH, or any group which can betransformed into an OH group, including a carbonyl. R₃ represents one ormore substituents independently selected from alkyl, alkoxyl, amino orhalogen groups. Alternatively, cleavable linkers may be constructed fromany labile functionality used on the 3′-block.

FIG. 3 is a schematic illustration of some of the 2′ or 3′ OH blockinggroups which can be present in the nucleotides or nucleosides accordingto the invention.

FIG. 4 shows two cycles of incorporation of a fully functional Tnucleoside triphosphate against a poly A template

DETAILED DESCRIPTION

The nucleotide or nucleoside molecules of the invention each have a basethat is linked to a detectable label via linkers that may be cleaved bycontact with water-soluble phosphines or water-soluble transitionmetal-containing catalysts described in greater detail hereinafter.Preferably moiety “T” is hydrogen. The base can be a purine, or apyrimidine. The base can be a deazapurine. The molecule can have aribose or deoxyribose sugar moiety. The ribose or deoxyribose sugar caninclude a protecting group attached via the 2′ or 3′ oxygen atom. Theprotecting group can be removed to expose a 3′-OH. The molecule can be adeoxyribonucleotide triphosphate. The detectable label can be afluorophore.

The invention also embraces oligonucleotides which comprise one or morenucleotides of the invention. Preferably, at least one nucleotide of theinvention is present at a terminal position in such aoligonucleotide.

The linker may be attached to the 5-position in pyrimidines or the7-position in purines or deazapurines. The characteristic feature of thenucleotides and nucleosides of the present invention is the amenabilityof the linkage to cleavage by certain water-soluble phosphines orphosphine-based transition metal catalysts. Since oligonucleotides aremanipulated in aqueous solution, the advantages of this water-solubilityare evident.

The cleavable linkages present in the nucleosides and nucleotides of theinvention each comprise an allyl or azido group.

Where the linkers comprise an azide-containing group the linkers maycontain a moiety of the formula:

These moieties may be present in either orientation in the linkerconnecting the base of the nucleotide/nucleoside with the detectablelabel, that is to say either of the bonds shown terminating in asterisksin each moiety may be closer to the base (or the label) in eachnucleotide or nucleoside than the other asterisk shown in eachstructure. Thus the invention embraces nucleotides and nucleoside havingschematically the following structures (shown on the left-hand side)which may be reacted with the water-soluble phosphines (described ingreater detail hereinafter) to generate the products shown on theright-hand side in which the azido-containing linker has been cleaved:

Whilst the connecting points * indicate the PG points at which themoieties are connected to the nucleotide or nucleoside, it will beappreciated that these points are generally the points at which themoiety is connected to the remainder of the linker. In other words, themoieties described herein that contain allyl or azido groups aregenerally part of, rather than exclusively constitute, the cleavablelinker.

Where the moiety is of formula —X—CH(N₃)—, the nature of thesubstituents to either side of the moiety —X—CH(N3)- affects thestability of the moiety and thus the rate at which it is cleaved. Forexample, where the linkage contains a substituted aryl group attached tothe moiety in which the substituents (indicated as “R” in the structuresimmediately below) are electron-withdrawing, this is manifested in theway in which the moiety cleaves. For example, electron-withdrawinggroups, serve to stabilise the linkage particularly where X is O or S.This makes cleavage occur more slowly.

Shown below schematically are the outcomes of four cleavages ofschematic nucleotide constructs and a further schematic representationof a preferred construct. The dotted lines connecting the “fluor”, dNTPor R group to the benzene ring or the cleavable moieties indicate thatsubstitution may be at any free position on the benzene ring and thatfurther atoms (not shown) in the linker may be present between thecleavable motif shown and the nucleotide and detectable label which thelinker connects:

Where the azide-containing moieties are Sieber linkers, i.e. are of theformula

cleavage of the moiety takes place across the bond linking the central6-membered ring of the tricycle to the amide such that a terminal amidegroup is left pendant to either the base or the fluorophore aftercleavage.

It will be appreciated that the azide-containing Sieber linker moietiesmay contain one or more substituents which may be eitherelectron-donating (examples include alkyl or alkoxy, e.g. C₁₋₆ alkyl orC₁₋₆ alkoxy groups) or electron-withdrawing groups (e.g. nitro, cyano,fluoro etc). Introduction of such substituents enables the skilledperson to tailor the conditions under which which cleavage may beeffected.

Where the nucleotides comprise an azide group in the linker, this may beconverted to the primary amine group with a thiol (in place of thephosphines), preferably a water-soluble thiol such as dithiothreitol(DTT).

Where the linkers comprise an allyl group, these may be of the formulae:

As with the azido-containing moieties discussed above, these linkingmoieties may be present in either orientation in the linker connectingthe base of the nucleotide/nucleoside with the detectable label.

Where the linkages comprise allyl-containing moieties, these linkers maybe cleaved with water-soluble transition metal catalysts formed from atransition metal and at least partially water-soluble ligands. Inaqueous solution these form at least partially water-soluble transitionmetal complexes.

The transition metal species serves to remove the allyl group. Where theallyl group is part of carbamate, deallylation affords the correspondingcarbamic acid. This spontaneously decarboxylates to afford a hemiaminalwhich hydrolyses so as to effect cleavage of the linker. Correspondingchemistries operate with the analogous thiocarbamate and carbonates soas to generate compounds containing moieties of structure *—C(NH₂)—X—*.These collapse by hydrolysing, again cleaving the linker.

By aqueous solution herein is meant a liquid comprising at least 20 vol%, preferably at least 50%, for example at least 75 vol %, particularlyat least 95 vol % and especially greater than above 98 vol %, ideally100 vol % of water as the continuous phase.

Transition metals of use in forming the catalysts described herein arepreferably platinum, palladium, rhodium, ruthenium, osmium and iridium.Palladium is particularly preferred.

The transition metal, e.g. palladium, is conveniently introduced as asalt, e.g. as a halide. Mixed salts such as Na₂PdCl₄ may also be used.Other appropriate salts and compounds will be readily determined by theskilled person and are commercially available, e.g. from AldrichChemical Company.

Suitable phosphines are any phosphine or mixed nitrogen-phosphineligands known to those skilled in the art, characterised in that theligands are derivatised so as to render them water-soluble, e.g. byintroducing one or more sulfonate, amine, hydroxyl (preferably aplurality of hydroxyl) or carboxylate residues. Where amine residues arepresent, formation of amine salts may assist the solublisation of theligand and thus the metal-allyl complex. Examples of appropriate ligandsare triaryl phosphines, e.g. triphenyl phosphine, derivatised so as tomake them water-soluble. Also preferred are trialkyl phosphines, e.g.tri-C₁₋₆-alkyl phosphines such as triethyl phosphines; such trialkylphosphines are likewise derivatised so as to make them water-soluble.Sulfonate-containing and carboxylate-containing phosphines areparticularly preferred; an example of the former3,3′,3″-phosphinidynetris (benzenesulfonic acid) which is commerciallyavailable from Aldrich Chemical Company as the trisodium salt; and apreferred example of the latter is tris(2-carboxyethyl)phosphine whichis available from Aldrich as the hydrochloride salt. The derivatisedwater-soluble phosphines and nitrogen-containing phosphines describedherein may be used as their salts (e.g. as the hydrochloride or sodiumsalts) or, for example, in the case of the sulfonic and carboxylicacid-containing phosphines described herein, as the free acids. Thus3,3′,3″-phosphinidynetris (benzenesulfonic acid) andtris(2-carboxyethyl)phosphines may be introduced either as the triacidsor the trisodium salts. Other appropriate salts will be evident to thoseskilled in the art. The existence in salt form is not particularlyimportant provided the phosphines are soluble in aqueous solution.

Other phosphines which may be used include the following:

The skilled person will be aware that the atoms chelated to thetransition metal in the water soluble complex may be part of mono- orpolydentate ligands. Some such polydentate ligands are shown above.Whilst monodentate ligands are preferred, the invention thus alsoembraces methods which use water-soluble bi-, tri-, tetra-, penta- andhexadentate water-soluble phosphine and water-solublenitrogen-containing phosphine ligands.

As noted earlier, the aqueous solution in which deprotection is effectedneed not be 100% (as the continuous phase). However, substantially purewater (e.g. at least 98 vol % and preferably about 100 vol %) ispreferred. Cosolvents are generally not required. Generally, nucleotidesand nucleosides are readily soluble in water (e.g. pure water) in whichthe linkage cleavage described herein may be effected. If desirable, oneor more water-miscible cosolvents may be employed. Appropriate solventsinclude acetonitrile or dimethylsulfoxide, methanol, ethanol andacetone, methanol being preferred. Less preferred solvents includetetrahydrofuran (THF) and dioxane.

In the methods of cleaving allyl-containing moieties according to theinvention, a soluble metal complex is formed comprising a transitionmetal and one or more water-soluble phosphine ligands (includingwater-soluble nitrogen-containing phosphine ligands). More than one typeof water-soluble phosphine/nitrogen-containing phosphine ligand may beused in any given reaction although generally only one type of theseclasses of ligand will be used in any given catalyst. The quantity oftransition metal, e.g. palladium, may be less than 1 mol % (calculatedrelative to the number of moles of linkage to be cleaved).Advantageously the amount of catalyst may be much less than 1 mol %,e.g. <0.50 mol %, preferably <0.10 mol %, particularly <0.05 mol %. Evenlower quantities of metal may be used, for example <0.03 or even <0.01mol %. As those skilled in the art will be aware, however, as quantityof catalyst is reduced, so too is the speed of the reaction. The skilledperson will be able to judge, in any instance, an appropriate quantityof transition metal and thus catalyst suitable for any particularreaction.

In contrast to the amount of metal required in forming the activecatalyst, the quantity of water-soluble phosphine-containing ligand(s)used is preferably be greater than 1 molar equivalent (again calculatedrelative to the number of moles of linkage to be cleaved). Preferablygreater than 4, e.g. greater than 6, for example 8-12 molar equivalentsof ligand may be used. Even higher quantities of ligand e.g. >20 moleequivalents may be used if desired.

The skilled person will be able to determine the quantity of ligand bestsuited to any individual reaction.

Where the linkage contains an azide group, the presence of a transitionmetal is not necessary to effect cleavage. Thus cleavage of such linkersmay be effected in the presence only of the phosphines discussed herein;these may be present in the methods of the invention as water-solublesalts, such as those discussed herein.

As is known in the art, a “nucleotide” consists of a nitrogenous base, asugar, and one or more phosphate groups. In RNA, the sugar is a ribose,and in DNA is a deoxyribose, i.e., a sugar lacking a hydroxyl group thatis present in ribose. The nitrogenous base is a derivative of purine orpyrimidine. The purines are adenosine (A) and guanidine (G), and thepyrimidines are cytidine (C) and thymidine (T) (or in the context ofRNA, uracil (U)). The C-1 atom of deoxyribose is bonded to N-1 of apyrimidine or N-9 of a purine. A nucleotide is also a phosphate ester ofa nucleoside, with esterification occurring on the hydroxyl groupattached to C-5 of the sugar. Nucleotides are usually mono, di- ortriphosphates.

A “nucleoside” is structurally similar to a nucleotide, but is missingthe phosphate moieties. An example of a nucleoside analog would be onein which the label is linked to the base and there is no phosphate groupattached to the sugar molecule.

Although the base is usually referred to as a purine or pyrimidine, theskilled person will appreciate that derivatives and analogs areavailable which do not alter the capability of the nucleotide ornucleoside to undergo Watson-Crick base pairing. “Derivative” or“analog” means a compound or molecule whose core structure is the sameas, or closely resembles that of, a parent compound, but which has achemical or physical modification, such as a different or additionalside group, which allows the derivative nucleotide or nucleoside to belinked to another molecule. For example, the base can be a deazapurine.

The modified nucleotides or nucleotides disclosed and claimed herein areexamples of such derivatives or analogues with the addition ofdetectable labels connected to the bases by the cleavable allyl- orazido-containing linkers described herein. Thus the terms nucleotidesand nucleosides as used herein will be understood to embrace suchmodified nucleosides and nucleotides.

The derivatives should be capable of undergoing Watson-Crick pairing.“Derivative” and “analogue” also mean a synthetic nucleotide ornucleoside derivative having modified base moieties and/or modifiedsugar moieties. Such derivatives and analogs are discussed in, e.g.,Scheit, Nucleotide Analogs (John Wiley & Son, 1980) and Uhlman et al.,Chemical Reviews 90:543-584, 1990. Nucleotide analogs can also comprisemodified phosphodiester linkages, including phosphorothioate,phosphorodithioate, alkyl-phosphonate, phosphoranilidate andphosphoramidate linkages. The analogs should be capable of undergoingWatson-Crick base pairing. “Derivative”, “analog” and “modified” as usedherein, may be used interchangeably, and are encompassed by the terms“nucleotide” and “nucleoside” defined herein.

The methods of the present invention make use of conventional detectablelabels. Detection can be carried out by any suitable method, includingfluorescence spectroscopy or by other optical means. The preferred labelis a fluorophore, which, after absorption of energy, emits radiation ata defined wavelength. Many suitable fluorescent labels are known. Forexample, Welch et al. (Chem. Eur. J. 5(3):951-960, 1999) disclosesdansyl-functionalised fluorescent moieties that can be used in thepresent invention. Zhu et al. (Cytometry 28:206-211, 1997) describes theuse of the fluorescent labels Cy3 and Cy5, which can also be used in thepresent invention. Labels suitable for use are also disclosed in Proberet al. (Science 238:336-341, 1987); Connell et al. (BioTechniques5(4):342-384, 1987), Ansorge et al. (Nucl. Acids Res. 15(11):4593-4602,1987) and Smith et al. (Nature 321:674, 1986). Other commerciallyavailable fluorescent labels include, but are not limited to,fluorescein, rhodamine (including TMR, Texas red and Rox), alexa,bodipy, acridine, coumarin, pyrene, benzanthracene and the cyanins.

Multiple labels can also be used in the invention. For example,bi-fluorophore FRET cassettes (Tet. Letts. 46:8867-8871, 2000) are wellknown in the art and can be utilised in the present invention.Multi-fluor dendrimeric systems (J. Amer. Chem. Soc. 123:8101-8108,2001) can also be used.

Although fluorescent labels are preferred, other forms of detectablelabels will be apparent as useful to those of ordinary skill. Forexample, microparticles, including quantum dots (Empodocles, et al.,Nature 399:126-130, 1999), gold nanoparticles (Reichert et al., Anal.Chem. 72:6025-6029, 2000), microbeads (Lacoste et al., Proc. Natl. Acad.Sci USA 97(17):9461-9466, 2000), and tags detectable by massspectrometry can all be used.

Multi-component labels can also be used in the invention. Amulti-component label is one which is dependent on the interaction witha further compound for detection. The most common multi-component labelused in biology is the biotin-streptavidin system. Biotin is used as thelabel attached to the nucleotide base. Streptavidin is then addedseparately to enable detection to occur. Other multi-component systemsare available. For example, dinitrophenol has a commercially availablefluorescent antibody that can be used for detection.

The label (or label and linker construct) can be of a size or structuresufficient to act as a block to the incorporation of a furthernucleotide onto the nucleotide of the invention. This permits controlledpolymerization to be carried out. The block can be due to sterichindrance, or can be due to a combination of size, charge and structure.

The invention will be further described primarily with reference tonucleotides. However, unless indicated otherwise, the references hereinto nucleotides are also intended to be applicable to nucleosides. Theinvention will also be further described with reference to DNA, althoughthe description will also be applicable to RNA, PNA, and other nucleicacids, unless otherwise indicated.

The modified nucleotides of the invention use a cleavable linker toattach the label to the nucleotide. The use of a cleavable linkerensures that the label can, if required, be removed after detection,avoiding any interfering signal with any labelled nucleotideincorporated subsequently.

The use of the term “cleavable linker” is not meant to imply that thewhole linker is required to be removed from the nucleotide base. Thecleavage site can be located at a position on the linker that ensuresthat part of the linker remains attached to the nucleotide base aftercleavage.

The linker can be attached at any position on the nucleotide baseprovided that Watson-Crick base pairing can still be carried out. In thecontext of purine bases, it is preferred if the linker is attached viathe 7-position of the purine or the preferred deazapurine analogue, viaan 8-modified purine, via an N-6 modified adenosine or an N-2 modifiedguanine. For pyrimidines, attachment is preferably via the 5-position oncytidine, thymidine or uracil and the N-4 position on cytosine. Suitablenucleotide structures are shown in FIG. 1. For each structure in FIG. 1,X can be H, phosphate, diphosphate or triphosphate. R₁ and R₂ can be thesame or different, and can be selected from H, OH, or any group whichcan be transformed into an OH, including, but not limited to, acarbonyl.

Suitable linkers comprise the azide- and allyl-containing moietiesdiscussed earlier. However, in addition to these cleavable moieties,other cleavable motifs may of course also be present in the linkers.

Referring to FIG. 2, examples of these include, but are not limited to,disulfide linkers (1), acid labile moieties (2, 3, 4 and 5; includingdialkoxybenzyl moieties (e.g., 2), Sieber linkers (e.g., 3), indolemoieties (e.g., 4), t-butyl Sieber moieties (e.g., 5)),electrophilically cleavable moieties, nucleophilically cleavablemoieties, photocleavable moieties, cleavage under reductive conditions,oxidative conditions, cleavage via use of safety-catch moieties, andcleavage by elimination mechanisms. Examples of such moieties aredescribed in WO03/048387.

As well as the moiety cleavable by water-soluble phosphines ortransition metal-based catalysts described herein, the cleavablelinkages may also comprise a spacer unit. The spacer distances thenucleotide base from the cleavage site or label. The length of themoiety is unimportant provided that the label is held a sufficientdistance from the nucleotide so as not to interfere with any interactionbetween the nucleotide and an enzyme.

Such spacing groups may contain one or more arylene, e.g. phenylene,groups in the chain (i.e. a moiety —Ar— where the phenyl ring is part ofthe linker by way of its 1,4, 1,3 or 1,2-disposed carbon atoms). Thephenyl ring may be substituted at its non-bonded position with one ormore substituents such as alkyl, hydroxyl, alkyloxy, halide, nitro,carboxyl or cyano and the like, particularly electron-withdrawinggroups, which electron-withdrawing is either by induction or resonance.An example of an electron-withdrawing group by resonance is nitro; agroup which acts through induction is fluoro. The skilled person will beaware of other appropriate electron-withdrawing groups. The linkage inthe R′ group may also include moieties such a —O—, —S(O)_(q), wherein qis 0, 1 or 2 or NH or Nalkyl.

The modified nucleotides can also comprise additional groups ormodifications to the sugar group. For example, a dideoxyribosederivative, lacking both hydroxyl groups on the ribose ring structure(at the 2′ and 3′ positions), can be prepared and used as a block tofurther nucleotide incorporation on a growing oligonucleotide strand.The protecting group is intended to prevent nucleotide incorporationonto a nascent polynucleotide strand, and can be removed under definedconditions to allow polymerisation to occur. In contrast to the priorart, there need be no detectable label attached at the ribose 3′position. This allows that steric hindrance with the polymerase enzymeto be reduced, while still allowing control of incorporation using theprotecting group.

The skilled person will appreciate how to attach a suitable protectinggroup to the ribose ring to block interactions with the 3′-OH. Theprotecting group can be attached directly at the 3′ position, or can beattached at the 2′ position (the protecting group being of sufficientsize or charge to block interactions at the 3′ position). Alternatively,the protecting group can be attached at both the 3′ and 2′ positions,and can be cleaved to expose the 3′OH group.

Suitable protecting groups will be apparent to the skilled person, andcan be formed from any suitable protecting group disclosed in“Protective Groups in Organic Synthesis”, T.W. Greene and P.G.M. Wuts,3rd Ed., Wiley Interscience, New York. The protecting group should beremovable (or modifiable) to produce a 3′ OH group. The process used toobtain the 3′ OH group can be any suitable chemical or enzymic reaction.

Preferably, the blocking, or protecting group is an allyl group or agroup of the structure

—O—Z

wherein Z is any of —C(R′)₂—O—R″, —C(R′)₂—N(R″)₂, —C(R′)₂—N(H)R″,—C(R′)₂—S—R″ and —C(R′)₂—F,

wherein each R″ is or is part of a removable protecting group;

each R′ is independently a hydrogen atom, an alkyl, substituted alkyl,arylalkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclic, acyl,cyano, alkoxy, aryloxy, heteroaryloxy or amido group; or (R′)₂represents an alkylidene group of formula —C(R′″)₂ wherein each R′″ maybe the same or different and is selected from the group comprisinghydrogen and halogen atoms and alkyl groups; and

wherein said molecule may be reacted to yield an intermediate in whicheach R″ is exchanged for H or, where Z is —C(R′)₂—F, the F is exchangedfor OH, SH or NH₂, preferably OH, which intermediate dissociates underaqueous conditions to afford a molecule with a free 3′OH;

with the proviso that where Z is —C(R′)₂—S—R″, both R′ groups are not H.

Where the blocking group is an allyl group, it may be introduced intothe 3′-position using standard literature procedures such as that usedby Metzker (Nucleic Acids Research, 22(20):4259-4267, 1994).

The intermediates produced advantageously spontaneously dissociate underaqueous conditions back to the natural 3′ hydroxy structure, whichpermits further incorporation of another nucleotide. Any appropriateprotecting group may be used. Preferably, Z is of formula —C(R′)₂—O—R″,—C(R′)₂—N(R″)₂, —C(R′)₂—N(H)R″ and —C(R′)₂—SR″. Particularly preferably,Z is of the formula —C(R′)₂—O—R″, —C(R′)₂—N(R″)₂, and —C(R′)₂—SR″. R″may be a benzyl group or a substituted benzyl group.

One example of groups of structure —O—Z wherein Z is —C(R′)₂—N(R″)₂ arethose in which —N(R″)₂ is azido (—N₃). One preferred such example isazidomethyl wherein each R′ is H. Alternatively, R′ in Z groups offormula —C(R′)₂—N₃ and other Z groups may be any of the other groupsdiscussed herein. Examples of typical R′ groups include C₁₋₆ alkyl,particularly methyl and ethyl, and the following (in which eachstructure shows the bond which connects the R′ moiety to the carbon atomto which it is attached in the Z groups; the asterisks (*) indicate; thepoints of attachment):

(wherein each R is an optionally substituted C₃₋₁₀ alkyl group, anoptionally substituted alkoxy group, a halogen atom or functional groupsuch as hydroxyl, amino, cyano, nitro, carboxyl and the like) and “Het”is a heterocyclic (which may for example be a heteroaryl group). TheseR′ groups shown above are preferred where the other R′ group is the sameas the first or is hydrogen. Preferred Z groups are of formula C(R′)₂N₃in which the R′ groups are selected from the structures given above andhydrogen; or in which (R′)₂ represents an alkylidene group of formula—C(R′″)₂, e.g. ═C(Me)₂.

Where molecules contain Z groups of formula C(R′)₂N₃, the azido groupmay be converted to amino by contacting such molecules with thephosphine or nitrogen-containing phosphines ligands described in detailin connection with the transition metal complexes which serve to cleavethe allyl groups from compounds of formula PN—O-allyl, formulaR—O-allyl, R₂N(allyl), RNH(allyl), RN(allyl)₂ and R—S-allyl. Whentransforming azido to amino, however, no transition metal is necessary.Alternatively, the azido group in Z groups of formula C(R′)₂N₃ may beconverted to amino by contacting such molecules with the thiols, inparticular water-soluble thiols such as dithiothreitol (DTT).

The labile linker may, and preferably does, consist of functionalitycleavable under identical conditions to the block. This makes thedeprotection process more efficient since only a single treatment willbe required to cleave both the label and the block. For example, wherethe linkage contains an allyl moiety as discussed and claimed herein andthe blocking group is an allyl group, both linkage and blocking groupwill be cleavable under identical conditions. Similarly, if the linkagecontains an azido moiety as discussed and claimed herein and theblocking group comprises an azido moiety, e.g. is of formula Z whereinR″ is N₃ as discussed hereinbefore, both linkage and blocking group willbe cleavable under identical conditions. The blocking group may ofcourse be deprotected under entirely different chemical conditions tothose required to cleave the linker.

The term “alkyl” covers both straight chain and branched chain alkylgroups. Unless the context indicates otherwise, the term “alkyl” refersto groups having 1 to 10 carbon atoms, for example 1 to 8 carbon atoms,and typically from 1 to 6 carbon atoms, for example from 1 to 4 carbonatoms. Examples of alkyl groups include methyl, ethyl, propyl,isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, 2-pentyl, 3-pentyl,2-methyl butyl, 3-methyl butyl, and n-hexyl and its isomers.

Examples of cycloalkyl groups are those having from 3 to 10 ring atoms,particular examples including those derived from cyclopropane,cyclobutane, cyclopentane, cyclohexane and cycloheptane, bicycloheptaneand decalin.

Where alkyl (including cycloalkyl) groups are substituted, particularlywhere these form either both of the R′ groups of the molecules of theinvention, examples of appropriate substituents include halogensubstituents or functional groups such as hydroxyl, amino, cyano, nitro,carboxyl and the like. Such groups may also be substituents, whereappropriate, of the other R′ groups in the molecules of the invention.

Examples of alkenyl groups include, but are not limited to, ethenyl(vinyl), 1-propenyl, 2-propenyl (allyl), isopropenyl, butenyl,buta-1,4-dienyl, pentenyl, and hexenyl.

Examples of cycloalkenyl groups include, but are not limited to,cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclopentadienyl andcyclohexenyl.

The term alkoxy refers to C₁₋₆ alkoxy unless otherwise indicated: —OR,wherein R is a C₁₋₆ alkyl group. Examples of C₁₋₆ alkoxy groups include,but are not limited to, —OMe (methoxy),—OEt (ethoxy), —O(nPr)(n-propoxy), —O(iPr) (isopropoxy), —O(nBu) (n-butoxy), —O(sBu)(sec-butoxy), —O(iBu) (isobutoxy), and —O(tBu) (tert-butoxy).

The term “halogen” as used herein includes fluorine, chlorine, bromineand iodine.

The nucleotide molecules of the present invention are suitable for usein many different methods where the detection of nucleotides isrequired.

DNA sequencing methods, such as those outlined in U.S. Pat. No.5,302,509 can be carried out using the nucleotide.

Preferably the blocked and labelled modified nucleotide constructs ofthe nucleotide bases A, T, C and G are recognised as substrates by thesame polymerase enzyme.

In the methods described herein, each of the nucleotides can be broughtinto contact with the target sequentially, with removal ofnon-incorporated nucleotides prior to addition of the next nucleotide,where detection and removal of the label and the blocking group, ifpresent is carried out either after addition of each nucleotide, orafter addition of all four nucleotides.

In the methods, all of the nucleotides can be brought into contact withthe target simultaneously, i.e., a composition comprising all of thedifferent nucleotides is brought into contact with the target, andnon-incorporated nucleotides are removed prior to detection andsubsequent to removal of the label and the blocking group, if present.

The four nucleotides, one of which will be complementary to the firstunpaired base in the target polynucleotide, may be brought into contactwith the target sequentially, optionally with removal ofnon-incorporated nucleotides prior to addition of the next nucleotide.Determination of the success of the incorporation may be carried outeither after provision of each nucleotide, or after the addition of allof the nucleotides added. If it is determined after addition of fewerthan four nucleotides that one has been incorporated, it is notnecessary to provide further nucleotides in order to detect thenucleotides complementary to the incorporated nucleotide.

Alternatively, all of the nucleotides can be brought into contact withthe target simultaneously, i.e., a composition comprising all of thedifferent nucleotide (i.e. A, T, C and G or A, U, C and G) is broughtinto contact with the target, and non-incorporated nucleotides removedprior to detection and removal of the label(s). The methods involvingsequential addition of nucleotides may comprise a first substep andoptionally one or more subsequent substeps. In the first substep acomposition comprising one, two or three of the four possiblenucleotides is provided, i.e. brought into contact with, the target.Thereafter any unincorporated nucleotides may be removed and a detectingstep may be conducted to determine whether one of the nucleotides hasbeen incorporated. If one has been incorporated, the cleavage of thelinker may be effected and, if necessary, a terminal amide functionalityintroduced thereafter to the pendant arm. In this way the identity of anucleotide in the target polynucleotide may be determined. The nascentpolynucleotide may then be extended to determine the identity of thenext unpaired nucleotide in the target oligonucleotide.

If the first substep above does not lead to incorporation of anucleotide, or if this is not known, since the presence of incorporatednucleotides is not sought immediately after the first substep, one ormore subsequent substeps may be conducted in which some or all of thosenucleotides not provided in the first substep are provided either, asappropriate, simultaneously or subsequently. Thereafter anyunincorporated nucleotides may be removed and a detecting step conductedto determine whether one of the classes of nucleotide has beenincorporated. If one has been incorporated, cleavage of the linker maybe effected, and if necessary as an additional step or steps, terminalamide functionality introduced to the pendant arm. In this way theidentity of a nucleotide in the target polynucleotide may be determined.The nascent polynucleotide may then be extended to determine theidentity of the next unpaired nucleotide in the target oligonucleotide.If necessary, a third and optionally a fourth substep may be effected ina similar manner to the second substep. Obviously, once four substepshave been effected, all four possible nucleotides will have beenprovided and one will have been incorporated.

It is desirable to determine whether a type or class of nucleotide hasbeen incorporated after any particular combination comprising one, twoor three nucleotides has been provided. In this way the unnecessary costand time expended in providing the other nucleotide(s) is obviated. Thisis not a required feature of the invention, however.

It is also desirable, where the method for sequencing comprises one ormore substeps, to remove any unincorporated nucleotides before furthernucleotide are provided. Again, this is not a required feature of theinvention. Obviously, it is necessary that at least some and preferablyas many as practicable of the unincorporated nucleotides are removedprior to the detection of the incorporated nucleotide.

A method for determining the sequence of a target polynucleotide can becarried out by contacting the target polynucleotide separately with thedifferent nucleotides to form the complement to that of the targetpolynucleotide, and detecting the incorporation of the nucleotides. Sucha method makes use of polymerisation, whereby a polymerase enzymeextends the complementary strand by incorporating the correct nucleotidecomplementary to that on the target. The polymerisation reaction alsorequires a specific primer to initiate polymerisation.

For each cycle, the incorporation of the modified nucleotide is carriedout by the polymerase enzyme, and the incorporation event is thendetermined. Many different polymerase enzymes exist, and it will beevident to the person of ordinary skill which is most appropriate touse. Preferred enzymes include DNA polymerase I, the Klenow fragment,DNA polymerase III, T4 or T7 DNA polymerase, Taq polymerase or Ventpolymerase. Polymerases engineered to have specific properties can alsobe used. Preferably, the molecule is incorporated by a polymerase andparticularly from Thermococcus sp., such as 90 N. Even more preferably,the polymerase is a mutant 90N A485L and even more preferably is adouble mutant Y409V and A485L. An example of one such preferred enzymeis Thermococcus sp. 90N exo —Y409V A485L available from New EnglandBiolabs. Examples of such appropriate polymerases are disclosed in Proc.Natl. Acad. Sci. USA, 1996(93), pp 5281-5285, Nucleic Acids Research,1999(27), pp 2454-2553 and Acids Research, 2002(30), pp 605-613.

Those skilled in the art will be aware of the utility ofdideoxynucleoside triphosphates in so-called Sanger sequencing methods,and related protocols (Sanger-type), which rely upon randomisedchain-termination at a particular type of nucleotide. An example of aSanger-type sequencing protocol is the BASS method described by Metzker(infra). Other Sanger-type sequencing methods will be known to thoseskilled in the art.

Sanger and Sanger-type methods generally operate by the conducting of anexperiment in which eight types of nucleotides are provided, four ofwhich contain a 3′OH group; and four of which omit the OH group andwhich are labeled differently from each other. The nucleotides usedwhich omit the 3′OH group-dideoxy nucleotides—are conventiallyabbreviated to ddNTPs. As is known by the skilled person, since theddNTPs are labeled differently, by determining the positions of theterminal nucleotides incorporated, and combining this information, thesequence of the target oligonucleotide may be determined.

It will be recognized that the nucleotides of the present invention inwhich the 3′OH group is either absent or blocked may be of utility inSanger methods and related protocols since the same effect achieved byusing ddNTPs may be achieved by using 3′-OH blocking groups: bothprevent incorporation of subsequent nucleotides.

The use of the nucleotides according to the present invention in Sangerand Sanger-type sequencing methods form a still further aspect of thisinvention. Viewed from this aspect, the invention provides the use ofsuch nucleotides in a Sanger or a Sanger-type sequencing method.

The sequencing methods are preferably carried out with the targetpolynucleotide arrayed on a solid support. Multiple targetpolynucleotides can be immobilised on the solid support through linkermolecules, or can be attached to particles, e.g., microspheres, whichcan also be attached to a solid support material.

The polynucleotides can be attached to the solid support by a number ofmeans, including the use of biotin-avidin interactions. Methods forimmobilizing polynucleotides on a solid support are well known in theart, and include lithographic techniques and “spotting” individualpolynucleotides in defined positions on a solid support. Suitable solidsupports are known in the art, and include glass slides and beads,ceramic and silicon surfaces and plastic materials. The support isusually a flat surface although microscopic beads (microspheres) canalso be used and can in turn be attached to another solid support byknown means. The microspheres can be of any suitable size, typically inthe range of from 10 nm to 100 nm in diameter. In a preferredembodiment, the polynucleotides are attached directly onto a planarsurface, preferably a planar glass surface. Attachment will preferablybe by means of a covalent linkage. Preferably, the arrays that are usedare single molecule arrays that comprise polynucleotides in distinctoptically resolvable areas, e.g., as disclosed in International App. No.WO 00/06770.

The sequencing method can be carried out on both single polynucleotidemolecule and multi-polynucleotide molecule arrays, i.e., arrays ofdistinct individual polynucleotide molecules and arrays of distinctregions comprising multiple copies of one individual polynucleotidemolecule. Single molecule arrays allow each individual polynucleotide tobe resolved separately. The use of single molecule arrays is preferred.Sequencing single molecule arrays non-destructively allows a spatiallyaddressable array to be formed.

The method makes use of the polymerisation reaction to generate thecomplementary sequence of the target. Conditions compatible withpolymerization reactions will be apparent to the skilled person.

To carry out the polymerase reaction it will usually be necessary tofirst anneal a primer sequence to the target polynucleotide, the primersequence being recognised by the polymerase enzyme and acting as aninitiation site for the subsequent extension of the complementarystrand. The primer sequence may be added as a separate component withrespect to the target polynucleotide. Alternatively, the primer and thetarget polynucleotide may each be part of one single stranded molecule,with the primer portion forming an intramolecular duplex with a part ofthe target, i.e., a hairpin loop structure. This structure may beimmobilised to the solid support at any point on the molecule. Otherconditions necessary for carrying out the polymerase reaction, includingtemperature, pH, buffer compositions etc., will be apparent to thoseskilled in the art.

The modified nucleotides of the invention are then brought into contactwith the target polynucleotide, to allow polymerisation to occur. Thenucleotides may be added sequentially, i.e., separate addition of eachnucleotide type (A, T, G or C), or added together. If they are addedtogether, it is preferable for each nucleotide type to be labelled witha different label.

This polymerisation step is allowed to proceed for a time sufficient toallow incorporation of a nucleotide.

Nucleotides that are not incorporated are then removed, for example, bysubjecting the array to a washing step, and detection of theincorporated labels may then be carried out.

Detection may be by conventional means, for example if the label is afluorescent moiety, detection of an incorporated base may be carried outby using a confocal scanning microscope to scan the surface of the arraywith a laser, to image a fluorophore bound directly to the incorporatedbase. Alternatively, a sensitive 2-D detector, such as a charge-coupleddetector (CCD), can be used to visualise the individual signalsgenerated. However, other techniques such as scanning near-field opticalmicroscopy (SNOM) are available and may be used when imaging densearrays. For example, using SNOM, individual polynucleotides may bedistinguished when separated by a distance of less than 100 nm, e.g., 10nm to 10 μm. For a description of scanning near-field opticalmicroscopy, see Moyer et al., Laser Focus World 29:10, 1993. Suitableapparatus used for imaging polynucleotide arrays are known and thetechnical set-up will be apparent to the skilled person.

After detection, the label may be removed using suitable conditions thatcleave the linker.

The use of the modified nucleotides is not limited to DNA sequencingtechniques, and other techniques, including polynucleotide synthesis,DNA hybridisation assays and single nucleotide polymorphism studies, mayalso be carried out using nucleotides of the invention. Any techniquethat involves the interaction between a nucleotide and an enzyme maymake use of the molecules of the invention. For example, the moleculemay be used as a substrate for a reverse transcriptase or terminaltransferase enzyme.

Suitable nucleotide structures are described in the followingnon-limiting Examples and are shown in the accompanying drawings.

3-(2,2-Diethoxy-ethoxy)-benzoic acid ethyl ester

2-Bromoacetaldehyde diethyl acetal (3 ml, 20 mmol),ethyl-3-hydroxy-benzoate (1.66 g, 10 mmol), potassium carbonate (2.76 g,20 mmol) and sodium iodide (0.298 g, 2 mmol) were heated at 120° C. indimethyl formamide (DMF) (15 ml) for 17 hrs. Another batch of2-bromoacetaldehyde diethyl acetal (3 ml, 20 mmol) was added and thereaction mixture was heated at 120° C. for another 24 hrs. The reactionwas cooled to room temperature and all the solvents were evaporatedunder reduced pressure. The residues were partitioned betweendichloromethane (DCM) (200 ml) and water (200 ml). The DCM layer wasseparated and the aqueous layer was back-extracted with DCM (2×100 ml).All the DCM extracts were combined, dried over MgSO₄ and evaporatedunder reduced pressure. The residue was purified by a columnchromatography (4×20 cm). The product was eluted with 100% DCM and thetitle compound was obtained as a colourless oil (2.21 g, 78.3%).

¹HNMR [CDCl₃]: 7.58 (1H, Ar—H, d J 7.7), 7.51 (1H, Ar—H, dd, J 2.4 and1.5), 7.27 (1H, Ar—H, t, J 8.0), 7.05 (1H, Ar—H, dd, J 7.9 and 2.3),4.79 (1H, CH, t, J 5.2), 4.20 (2H, OCH₂, q, J 7.2), 3.99 (2H, ArOCH₂, d,J 5.2), 3.75-3.65 (2H, OCH₂, m), 3.63-3.53 (2H, OCH₂, m), 1.33 (3H, CH₃,t, J 7.2) and 1.19 (6H, CH₃, t, J 7.1).

3-(2-Azido-2-ethoxy-ethoxy)-benzoic acid ethyl ester

To a mixture of 3-(2,2-diethoxy-ethoxy)-benzoic acid ethyl ester (1.128g, 4 mmol) and azidotrimethylsilane (0.584 ml, 4.4 mmol) was added SnCl₄(40 μl) at room temperature. After 1 hr, the precipitates were filteredoff and the filtrate was evaporated under reduced pressure. The residuewas dissolved in methanol, after 10 minutes, solvent was removed underreduced pressure. The residue was purified by a column chromatography(3×20 cm). The product was eluted with 10% petroleum ether (60-80° C.)in DCM. The title compound was obtained as a colourless oil (0.909 g,81.5%).

¹HNMR [CDCl₃]: 7.51 (1H, Ar—H, d J 7.7), 7.41 (1H, Ar—H, dd, J 2.6 and1.5), 7.19 (1H, Ar—H, t, J 7.9), 6.96 (1H, Ar—H, ddd, J 8.2, 2.7 and0.9), 4.63 (1H, CHN₃, t, J 5.1), 4.20 (2H, OCH₂, q, J 7.2), 4.05-3.90(2H, ArOCH₂, m), 3.80-3.78 (1H, OCH₂, H_(a), m), 3.55-3.47 (1H, OCH₂,H_(b), m), 1.22 (3H, CH₃, t, J 7.1) and 1.13 (3H, CH₃, t, J 7.1).

3-(2-Azido-2-ethoxy-ethoxy)-benzoic acid

3-(2,2-Diethoxy-ethoxy)-benzoic acid ethyl ester (0.279 g, 1 mmol) wasstirred with 4 M aqueous sodium hydroxide (2.5 ml, 10 mmol) and ethanol(2.5 ml) at room temperature. After 4 hrs, all the solvents were removedunder reduced pressure and the residue was dissolved in 50 ml water. Thesolution was acidified with 1 N HCl to pH 2 and then extracted with DCM(3×50 ml). All the DCM extracts were combined, dried over MgSO₄ andevaporated under reduced pressure. The title compound was obtained as acolourless solid (0.247 g, 98.4%).

¹HNMR [CDCl₃]: 7.68 (1H, Ar—H, d J 7.7), 7.58 (1H, Ar—H, dd, J 2.5 and1.5), 7.34 (1H, Ar—H, t, J 8.0), 7.13 (1H, Ar—H, dd, J 7.4 and 2.7),4.74 (1H, CHN₃, t, J 5.0), 4.20-4.02 (2H, ArOCH₂, m), 3.95-3.80 (1H,OCH₂, H_(a), m), 3.70-3.55 (1H, OCH₂, H_(b), m) and 1.24 (3H, CH₃, t, J7.1).

Triethyl ammonium salt of phosphoric acidmono-[5-(5-{3-[3-(2-azido-2-ethoxy-ethoxy)-benzoylamino-prop-1-ynyl}-2,4-dioxo-3,4-dihydro-2K-pyrimidin-1-yl)-3-hydroxy-tetrahydro-furan-2-yl]ester

3-(2,2-Diethoxy-ethoxy)-benzoic acid (3.77 mg, 15. μmol) was stirredwith N,N′-disuccinimidyl carbonate (3.84 mg, 15 μmol) and4-dimethylaminopyridine(DMAP) (1.83 mg, 15 μmol) in dry DMF (1 ml) atroom temperature. After 15 minutes, all the reaction mixture was addedto a solution of the triethyl ammonium salt of phosphoric acidmono-{5-[5-(3-amino-prop-1-ynyl)-2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-yl]-3-hydroxy-tetrahydro-furan-2-yl}ester (5 μmol) in 0.1 M carbonate buffer (0.1 M NaHC₃/0.1 M Na₂CO₃, v/v,1:1) (0.5 ml). After 5 hrs at room temperature, the reaction was dilutedwith 0.05 M triethylammonium bicarbonate (TEAB) buffer (TEAB, pH 7.5)(10 ml). The resulting solution was applied onto a short column of DEAEA-25 (1×5 cm). The column was initially eluted with 0.1 M TEAB buffer(50 ml) and then 0.7 M buffer (50 ml). The 0.7 M TEAB eluents werecollected and evaporated under reduced pressure. The residue wasco-evaporated with MeOH (2×10 ml) and then purified by preparative HPLC[HPLC gradient: A, 100% 0.1 M TEAB; B 100% MeCN; 0-2 min, 5% B (flow2-10 ml/min); 2-19 min, 5-40% B (flow 10 ml/min); 19-21 min, 40-95% B(flow 10 ml/min); 21-24 min, 95% B (flow 10 ml/min); 24-26 min, 95-5% B(flow 10 ml/min); 26-30 min, 5% B (flow 10-2 ml/min)]. The product withretention time of 19.6 min was collected and evaporated under reducedpressure and the residue was co-evaporated with methanol (3×5 ml) togive the title compound as triethyl ammonium salt (3.67 mg, yield 80%).¹HNMR in D₂O indicated approximately 3.2 triethylammonium count ions.

¹HNMR [D₂O]: 7.89 (1H, H-6, s), 7.44-7.31 (3H, Ar—H, m), 7.28 (1H, Ar—H,m), 7.12 (1H, Ar—H, dt, J 7.1 and 2.3), 6.17 (1H, H-1′, t, J 6.9), 4.96(1H, CHN₃, t, J 4.4), 4.43-4.35 (1H, H-3′, m), 4.2$ (2H, CHN, s),4.18-4.06 (2H, H-5′, m), 4.05-3.90 (1H, H-4′, m), 3.89-3.50 (4H, ArOCH₂,OCH₂, m), 3.03 (19H, CH₂N, q, J 7.3), 2.25-2.15 (2H, CH₂, m), 1.13 (32H,CH₃, t, J 7.3). ³¹P [D₂O]: 5.17 (s). MS-ES(−), m/z 593 [M−1].

2-(1-Azido-ethoxy)-ethanol

To a mixture of 2-methyl dioxolane (0.897 ml, 10 mmol) andazidotrimethylsilane (1.6 ml, 12 mmol) was added SnCl₄ (40 μl) at −78°C. After addition, the cooling bath was removed and the reaction waswarmed up to room temperature. After 1 hr, the reaction was worked up bypartitioning between DCM (50 ml) and saturated aqueous NaHCO₃ (50 ml).The aqueous layer was further extracted with DCM (20 ml). All the DCMextracts were combined, dried over MgSO₄ and evaporated under reducedpressure. The residue was dissolved in 10% aqueous methanol. After 2 hrat room temperature, all the solvents were removed under reducedpressure. The title compound was obtained as a colourless oil (224 mg,17.1%).

¹HNMR [CDCl₃]: 4.46 (1H, CHN₃, q, J 5.7), 3.77-3.68 (1H, OCH₂, H_(a),m), 3.63 (2H, OCH₂, t, J 4.3), 3.48-3.40 (1H, OCH₂, H_(b), m) and 1.34(3H, CH₃, d, J 5.7).

[2-(1-Azido-ethoxy)-ethoxy]-acetic acid ethyl ester

To a solution of 2-(1-azido-ethoxy)-ethanol (0.15 g, 1.14 mmol) in drytetrahydrofuran (THF) (5 ml) was added NaH (60% dispersion, 0.08 g, 2mmol) at 0° C. After 15 minutes, ethyl-2-bromoacetate (0.222 ml, 2 mmol)was added. The reaction was maintained at this temperature for 15minutes and then warmed up to room temperature. The reaction wasquenched by addition of saturated aqueous NaHCO₃ (5 ml) after 1 hr.After a further period of 5 min, the mixture was partitioned between DCM(50 ml) and saturated aqueous NaHCO₃ (50 ml). The aqueous layer wasfurther extracted with DCM (50 ml). All the DCM extracts were combined,dried over MgSO₄ and evaporated under reduced pressure. The titlecompound was obtained as an oil (0.162 g, 65.5%).

¹HNMR [CDCl₃]: 4.51 (1H, CHN₃, q, J 5.6), 4.10 (2H, OCH₂, q, J 7.1),4.03 (2H, OCH₂C(O), s), 3.85-3.77 (1H, OCH₂, H_(a), m), 3.67-3.57 (3H,OCH₂, H_(b), OCH₂, m), 1.37 (3H, CH₃, d, J 5.7) and 1.17 (3H, CH₃, t, J7.1).

[2-(1-Azido-ethoxy)-ethoxy]-acetic acid

[2-(1-Azido-ethoxy]-ethoxy-acetic acid ethyl ester (0.10 g, 0.46 mmol)was stirred with 4 M aqueous sodium hydroxide (1.15 ml, 4.6 mmol) andethanol (1.15 ml) at room temperature. After 4 hrs, all the solventswere removed under reduced pressure and the residue was dissolved in 5ml water. The solution was adjusted to pH 5 with 1 N KH₂PO₄ and thenextracted with DCM (2×15 ml). The aqueous layer was then acidified with1 N HCl to pH 2 and then extracted with DCM (3×25 ml). All the DCMextracts were combined, dried over MgSO₄ and evaporated under reducedpressure. The title compound was obtained as a colourless solid (42 mg,48.3%).

¹HNMR [CDCl₃]: 4.55 (1H, CHN₃, q, J 5.7), 4.15 (2H, OCH₂C(O), s),3.92-3.84 (1H, OCH₂, H, m), 3.74-3.70 (2H, OCH₂, m), 3.68-3.57 (1H,OCH₂, H_(b), m) and 1.44 (3H, CH₃, d, J 5.7).

Triethyl ammonium salt of phosphoric acidmono-{5-[5-(3-{2-[2-(1-azido-ethoxy)-ethoxy]-acetylamino}-prop-1-ynyl)-2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-yl]-3-hydroxy-tetrahydro-furan-2-yl}ester

[2-(1-Azido-ethoxy)-ethoxy]-acetic acid (5.7 mg, 30 μmol) was stirredwith N,N′-disuccinimidyl carbonate (7.68 mg, 30 μmol) and DMAP (3.7 mg,30 μmol) in dry DMF (2 ml) at room temperature. After 10 minutes, allthe reaction mixture was added to a solution of the triethyl ammoniumsalt of phosphoric acidmono-{5-[5-(3-amino-prop-1-ynyl)-2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-yl]-3-hydroxy-tetrahydro-furan-2-yl}ester (10 μmol) in 0.1 M TEAB (0.5 ml). After 5 hrs at room temperature,the reaction was diluted with 0.05 M triethylammonium bicarbonate buffer(TEAB, pH 7.5) (10 ml). The resulting solution was applied onto a shortcolumn of DEAE A-25 (1×10 cm). The column was initially eluted with 0.1M TEAB buffer (50 ml) and then 0.5 M buffer (50 ml). The 0.5 M TEABeluents were collected and evaporated under reduced pressure. Theresidue was co-evaporated with MeOH (2×10 ml) and then purified bypreparative HPLC [HPLC gradient: A, 100% 0.1 M TEAB; B 100% MeCN; 0-2min, 5% B (flow 2-10 ml/min); 2-15 min, 5-15% B (flow 10 ml/min); 15-28min, 15-22% B (flow 10 ml/min); 28-30 min, 22-95% B (flow 10 ml/min);30-34 min, 95% B (flow 10 ml/min); 34-36 min, 95-5% B (flow 10 ml/min);36-40 min, 5% B (flow 10-2 ml/min)]. The product with retention time of21.3 min was collected and evaporated under reduced pressure and theresidue was co-evaporated with methanol (3×5 ml) to give the titlecompound as triethyl ammonium salt (9.3 mmol, quantification at λ₂₈₈ in0.1 M TEAB buffer, 93%). ¹HNMR in D₂O indicated approximately fivetriethylammonium count ions.

¹HNMR [D₂O]: 7.85 (1H, H-6, s), 6.15 (1H, H-1′, t, J 6.8), 4.75 (1H,CHN₃, q, J 5.7), 4.38 (1H, H-3′, m), 4.09 (2H, OCH₂C(O), s), 3.99 (2H,CHN, s), 3.95 (1H, H-4′, m), 3.86-3.60 (6H, OCH₂CH₂O, H-5′, m), 3.03(30H, CH₂N, q, J 7.2), 2.22-2.12 (2H, CH₂, m), 1.31 (3H, CH₃, d, J 5.7)and 1.11 (45H, CH₃, t, J 7.2). ³¹P [D₂O]5.14 (s). MS-ES(−), m/z, 531[M−1].

3-([1,3]Dioxolan-2-ylmethoxy)-benzoic acid ethyl ester

2-Bromomethyl-1, 3-dioxolane (8.3 ml, 80 mmol), ethyl-3-hydroxy-benzoate(3.32 g, 20 mmol), potassium carbonate (5.53 g, 40 mmol) and sodiumiodide (1.2 g, 8 mmol) were heated at 120° C. in DMF (8 ml) for 17 hrs.The reaction was cooled to room temperature and all the solvents wereevaporated under reduced pressure. The residues were partitioned betweenDCM (250 ml) and water (250 ml). The DCM layer was separated and theaqueous layer was back-extracted with DCM (2×100 ml). All the DCMextracts were combined, dried over MgSO₄ and evaporated under reducedpressure. The residue was purified by column chromatography (4×25 cm).The product was eluted with 20% petroleum ether (60-80° C.) in DCM andthe title compound was obtained as a slightly brown oil (4.63 g, 91.8%).APCI-MS, m/z 252.95 (M+1).

¹HNMR [CDCl₃]: 1.39 (3H, CH₃, t, J 7.2), 3.96-4.09 (6H, OCH₂CH₂O,ArOCH₂, m), 4.36 (2H, OCH₂, q, J 7.2), 5.31 (1H, CH, t, J 4.0), 7.14(1H, Ar—H, ddd, J 1.6, 2.6 and 8.2), 7.34 (1H, Ar—H, t, J 7.9), 7.59(1H, Ar—H, dd, J 1.5 and 2.5) and 7.67 (1H, Ar—H, dt, J 1.4 and 7.6).

3-[2-Azido-2-(2-hydroxy-ethoxy)-ethoxy]-benzoic acid ethyl ester

To a mixture of 3-([1, 3]-dioxolan-2-ylmethoxy)-benzoic acid ethyl ester(2.02 g, 8 mmol) and azidotrimethylsilane (1.17 ml, 8.8 mmol) was addedSnCl₄ (60 μl) at room temperature under nitrogen. After 2 hr, 2% aqueousmethanol (10 ml) was added to the reaction mixture and the reaction wasstirred at room temperature for 30 minutes. All the solvents wereevaporated under reduced pressure. The residue was co-evaporated withethanol (2×10 ml). The residue was purified by a column chromatography(3×20 cm). The product was eluted with 0 to 1% methanol in DCM. Thetitle compound was obtained as a colourless oil (2.01 g, 85.1%).APCI-MS, m/z 267.90 (M-N₂+1).

¹HNMR [CDCl₃]: 1.38 (3H, CH₃, t, J 7.1), 3.73-3.86 (3H, OCH₂, H_(a),OCH₂, m), 3.99-4.05 (1H, OCH₂, H_(b), m), 4.17 (1H, Ar—OCH₂, H_(a), dd,J 4.9 and 10.1), 4.23 (1H, ArOCH₂, H_(b), dd, J 5.2 and 10.1), 4.38 (2H,OCH₂, q, J 7.1), 4.89 (1H, CH—N₃, t, J 5.1), 7.13 (1H, Ar—H, dd, J 2.1and 8.4), 7.36 (1H, Ar—H, t, J 7.9), 7.60 (1H, Ar—H, dd, J 1.0 and 2.5)and 7.70 (1H, Ar—H, d, J 7.8).

3-[2-Azido-2-(2-hydroxy-ethoxy)-ethoxy]-benzoic acid

3-[2-Azido-2-(2-hydroxy-ethoxy)-ethoxy]-benzoic acid ethyl ester (1.34g, 4.55 mmol) was stirred with 4 M aqueous sodium hydroxide (11.4 ml,45.5 mmol) and ethanol (11.4 ml) at room temperature. After 3 hrs, allthe solvents were removed under reduced pressure and the residue wasdissolved in 50 ml water. The solution was acidified with 1 N HCl to pH2 and then extracted with DCM (3×50 ml). All the DCM extracts werecombined, dried over MgSO₄ and evaporated under reduced pressure. Thetitle compound was obtained as a colourless solid (1.2 g, 98.7%). ES-MS,m/z 265.85 (M−1).

¹HNMR [CDCl₃]: 3.75-3.90 (3H, OCH₂, H_(a), OCH₂, m), 4.00-4.08 (1H,OCH₂, H_(b), m), 4.17 (1H, Ar—OCH₂, H_(a), dd, J 4.8 and 10.1), 4.24(1H, ArOCH₂, H_(b), dd, J 5.1 and 10.1), 4.90 (1H, CH—N₃, t, J 5.1),7.19 (1H, Ar—H, dd, J 2.5 and 8.2), 7.40 (1H, Ar—H, t, J 8.0), 7.60 (1H,Ar—H, s) and 7.70 (1H, Ar—H, d, J 7.9).

3-[2-Azido-2-(2-ethoxycarbonylmethoxy-ethoxy)-ethoxy]-benzoic acid

To a solution of 3-[2-azido-2-(2-hydroxy-ethoxy)-ethoxy]-benzoic acid(0.535 g, 2 mmol) in dry THF (6 ml) was added NaH (60% dispersion, 0.246g, 6 mmol) at 0° C. After 10 minutes, ethyl-2-bromoacetate (0.488 ml,4.4 mmol) was added. The reaction was then warmed up to room temperatureand stirred for 4 hours. The reaction was quenched by pouring it intoice-cold water (50 ml). The mixture was extracted with DCM (2×50 ml) andthe DCM extracts were discarded. The aqueous layer was then acidified topH 2 with 1 N HCl, and extracted with DCM (2×50 ml). These DCM extractswere combined, dried over MgSO₄ and evaporated under reduced pressure.The residue was purified by column chromatography (1×20 cm). The titlecompound, eluted with 2% methanol in DCM, was obtained as an oil (0.223g, 31.6%). ES-MS, m/z 351.95 (M−1).

¹HNMR [CDCl₃]: 1.29 (3H, CH₃, t, J 7.2), 3.81 (2H, OCH₂, t, J 4.4), 3.90(1H, OCH₂, H_(a), m), 4.04 (1H, OCH₂, H_(b), m), 4.13-4.27 (6H, ArOCH₂,OCH₂ and OCH₂C(O)), 4.95 (1H, CH—N₃, m), 7.19 (1H, Ar—H, dd, J 1.7 and8.3), 7.40 (1H, Ar—H, t, J 7.8), 7.63 (1H, Ar—H, s) and 7.76 (1H, Ar—H,d, J 7.6).

[2-(1-Azido-2-(3-[2-(2,2,2-trifluoroacetylamino)-ethylcarbamoyl]-phenoxy)-ethoxy)-ethoxy]-aceticacid ethyl ester

3-[2-Azido-2-(2-ethoxycarbonylmethoxy-ethoxy)-ethoxy]-benzoic acid(0.212 g, 0.6 mmol) was stirred with N,N′-disuccinimidyl carbonate(0.184 g, 0.72 mmol) and DMAP (0.088 g, 0.72 mmol) in dry DMF (1 ml) atroom temperature. After 10 minutes, trifluoroacetic acid salt ofN-(2-amino-ethyl)-2,2,2-trifluoro-acetamide (0.194 g, 0.72 mmol) wasadded followed by diisopropylethylamine (DIPEA) (0.251 ml, 1.44 mmol).The reaction mixture was then stirred at room temperature for 17 hours.All the solvents were evaporated under reduced pressure and the residueswere partitioned between DCM (50 ml) and aqueous NaH₂PO₄ (1 N, 50 ml).The aqueous layer was further extracted with DCM (2×25 ml). All the DCMextracts were combined, dried over MgSO₄ and evaporated under reducedpressure. The residue was purified by column chromatography (1×20 cm).The title compound, eluted with 1% methanol in DCM, was obtained as anoil (0.255 g, 86.6%). ES-MS, m/z 490.10 (M−1).

¹HNMR [CDCl₃]: 1.28 (3H, CH₃, t, J 7.1), 3.60 (2H, CH₂N, m), 3.69 (2H,CH₂N, m), 3.80 (2H, OCH₂, t, J 4.3), 3.86 (1H, OCH₂, H_(a), m), 4.04(1H, OCH₂, H_(b), m), 4.10-4.25 (6H, ArOCH₂, OCH₂ and OCH₂C (O), m),4.92 (1H, CH—N₃, m), 6.85 (1H, NH, br), 7.09 (1H, Ar—H, m), 7.37 (3H,Ar—H, m), and 7.96 (1H, NH, br).

(2-{2-[3-(2-Amino-ethylcarbamoyl)-phenoxy]-1-azido-ethoxy}-ethoxy)-aceticacid

[2-(1-Azido-2-{3-[2-(2,2,2-trifluoroacetylamino)-ethoxycarbamoyl]-phenoxy}-ethoxy)-ethoxy]-aceticacid ethyl ester (0.196 g, 0.4 mmol) was stirred with 4 M aqueous sodiumhydroxide (1 ml, 4 mmol) and ethanol (1 ml) at room temperature. After 2hrs, all the solvents were removed under reduced pressure and theresidue was dissolved in 15 ml water. The solution was extracted withDCM (2×15 ml). The DCM extracts were discarded and the aqueous layer wasacidified with 1 N HCl to pH 2. Then the solution was extracted againwith DCM (3×15 ml). The DCM extracts were discarded and the aqueouslayer was neutralised with 1 N NaOH to pH 8 and then evaporated underreduced pressure to dryness. The white solids were triturated withDCM/MeOH (v/v; 1:1, 2×25 ml). All the solids were filtered off and thefiltrates were combined and evaporated under reduced pressure to give agum. The gum was added in 10% MeOH in DCM (15 ml) and the insoluble,white solids were filtered off. The filtrates were evaporated underreduced pressure to give the mono-sodium salt of the title compound as acolourless powder (0.135 g, 86.6%). ES-MS, m/z 368.00 (M+1).

¹HNMR [D₂O]: 3.01 (2H, CH₂NH₂, t, J 6.0), 3.51 (2H, CH₂N, t, J 6.0),3.62 (2H, OCH₂, m), 3.77 (1H, OCH₂, H_(a), m), 3.80 (2H, CH₂C(O), s),3.96 (1H, OCH₂, H_(b), m), 4.19 (2H, ArOCH₂, d, J 4.3), 5.01 (1H, CH—N₃,t, J 4.5), 7.13 (1H, Ar—H, d, J 7.9) and 7.25-7.39 (3H, Ar—H, m).

[2-(1-Azido-2-{3-[2-(6-Cy3-hexanoylamino)-ethylcarbamoyl]-phenoxy}-ethoxy)-ethoxy]-aceticacid

The commercial Cy3 mono N-hydroxysuccinimide ester (5 mg, 6.53 μmol) and(2-{2-[3-(2-amino-ethylcarbamoyl)-phenoxy]-1-azido-ethoxy}-ethoxy)-aceticacid (7.2 mg, 19.6 μmol) were stirred together in dry DMF. DIPEA (6.82μl, 39.2 μmol) was added. After 2 hr stirring at room temperature, allthe solvent was evaporated under reduced pressure. The residue waspurified by preparative HPLC [HPLC gradient: A, 100% 0.1 M TEAB; B 100%MeCN; 0-2 min, 5% B (flow 2-10 ml/min); 2-19 min, 5-45% B (flow 10ml/min); 19-21 min, 45-95% B (flow 10 ml/min); 21-24 min, 95% B (flow 10ml/min); 24-26 min, 95-5% B (flow 10 ml/min); 26-30 min, 5% B (flow 10-2ml/min)]. The title compound with retention time of 17.85 min wasobtained as a pink solid (4.93 μmol, 75.5%, quantification at 550 nm inwater). ES-MS, m/z 488.95 [(M/2)-1]. ¹HNMR in D₂O indicatedapproximately 1.8 triethylammonium count ions.

¹HNMR [D₂O]: 1.15 (16.2H, CH₃ (Et₃N), t, J 7.2), 1.21 (3H, CH₃, t, J7.1), 1.47 (2H, CH₂, m), 1.55 (2H, CH₂, m), 1.58 (6H, 2×CH₃, s), 1.61(6H, 2×CH₃, s), 2.10 (2H, CH₂C(O), t, J 6.5), 3.06 (10.8H, CH₂ (Et₃N),q, J 7.2), 3.23 (2H, CH₂N, t, J 5.5), 3.32 (2H, CH₂N, t, J 5.8), 3.56(2H, OCH₂, m), 3.67-3.78 (3H, OCH₂, H_(a) and CH₂N, m), 3.79 (2H,OCH₂C(O), s), 3.85-3.97 (3H, OCH₂, H_(b) and CH₂N, m), 3.98 (2H, ArOCH₂,d, J 4.4), 4.85 (1H, CH—N₃, t, J 4.3), 6.14 (1H, ═CH, d, J 13.4), 6.19(1H, —CH, d, J 13.4), 6.90 (1H, Ar—H, m), 7.10-7.19 (5H, Ar—H, m), 7.69(2H, Ar—H, d, J 8.4), 7.73 (1H, Ar—H, s), 7.77 (1H, Ar—H, s) and 8.36(1H, —CH, t, J 13.4).

5-[3-(-Cy3-azidolinkeracetylamino)-prop-1-ynyl]-3′-azidomethoxy-dUTP

[2-(1-Azido-2-{3-[2-(6-Cy3-hexanoylamino)-ethylcarbamoyl]-phenoxy}-ethoxy)-ethoxy]-aceticacid (2 μmol) was stirred with N,N′-disuccinimidyl carbonate (0.563 mg,2.2 μmol) and DMAP (0.269 mg, 2.2 μmol) in dry DMF (1 ml) at roomtemperature. After 10 minutes, all the reaction mixture was added to asolution of the tri-n-butyl ammonium salt of[5-(3-amino-prop-1-ynyl)]-3′-azidomethoxy-dUTP (6 μmol, prepared byevaporating an aqueous solution of[5-(3-amino-prop-1-ynyl)]-3′-azidomethoxy-dUTP with tri-n-butyl amine(72 μl, 300 μmol)). The reaction mixture was sonicated for 5 minutes,then stirred at room temperature for 3 hrs. The reaction mixture wasdiluted with chilled 0.1 M TEAB (10 ml), the resulting solution wasapplied onto a short column of DEAE A-25 (1×10 cm). The column wasinitially eluted with 0.1 M TEAB buffer (50 ml) and then 1.0 M buffer(50 ml). The 1.0 M TEAB eluates were collected and evaporated underreduced pressure. The residue was co-evaporated with MeOH (2×10 ml) andthen purified by semi-preparative HPLC [HPLC gradient: A, 100% 0.1 MTEAB; B 100% MeCN; 0-2 min, 5% B (flow 2-5 ml/min); 2-14 min, 5-20% B(flow 5 ml/min); 14-20 min, 20-23% B (flow 5 ml/min); 20-22 min, 23-95%B (flow 5 ml/min); 22-25 min, 95% B (flow 5 ml/min); 25-26 min, 95-5% B(flow 5 ml/min); 26-30 min, 5% B (flow 5-2 ml/min)]. The product withretention time of 19.2 min was collected and evaporated under reducedpressure and the residue was co-evaporated with methanol (3×5 ml) togive the title compound as triethyl ammonium salt (1.22 μmol,quantification at λ₅₅₀ in 0.1 M TEAB buffer, 61.1%). ¹HNMR in D₂Oindicated approximately seven triethylammonium count ions.

¹HNMR [D₂O]: 1.14 (63H, CH₃ (Et₃N), t, J 7.3), 1.23 (3H, CH₃, t, J 7.1),1.49 (2H, CH₂, m), 1.55 (2H, CH₂, m), 1.60 (6H, 2×CH₃, s), 1.62 (6H,2×CH₃, s), 1.95-2.07 (1H, H_(a)-2′, m), 2.13 (2H, CH₂C(O), t, J 6.7),2.17-2.27 (1H, H_(b)-2′, m), 3.05 (42H, CH₂ (Et₃N), q, J 7.3), 3.27 (2H,CH₂N, t, J 5.7), 3.37 (2H, CH₂N, t, J 5.8), 3.55-3.79 (5H, m), 3.80-4.10(11H, m), 4.15 (1H, H-4′, m), 4.38 (1H, H-3′, m), 4.80 (2H, OCH2-N₃, s),4.88 (1H, CH—N₃, m), 5.95 (1H, H-1′, q, J 7.6), 6.13 (1H, ═CH, d, J13.4), 6.20 (1H, —CH, d, J 13.4), 6.84 (1H, Ar—H, d, J 7.3), 7.05-7.24(5H, Ar—H, m), 7.60-7.80 (5H, Ar—H and H-6, m) and 8.35 (1H, ═CH, t, J13.4). ³¹PNMR [D₂O]: −20.82 (PP, m), −10.07 (^(α)P, d, J 16.2) and −4.90(^(γ)P, d, J 18.9).

2 Cycles of Fully Functional Nucleoside Triphosphate (FFN) Preparationof Beads

Take 15 μL of Dynabeads® M-280 streptavidin coated beads (DynalBiotech), remove storage buffer and wash 3 times with 150 μL of TEbuffer (Tris.HCl pH 8, 10 mM and EDTA, 1 mM). Resuspend in 37.5 μL of B& W buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA and 2.0 M NaCl), add 10 μLof biotinylated ³²P labelled hairpin DNA and add 27.5 μL of water. Allowto stand at room temperature for 15 minutes. Remove buffer and washbeads 3 times with 100 μL of TE buffer.

Incorporation of the 1^(st) FFN

75 μL reaction, Tris.HCl pH 8.8 50 mM, Tween-20, 0.01%, MgSO₄ 4 mM,MnCl₂, 0.2 mM, add 2 M FFN and 100 nM polymerase (Thermococcus sp. 9° Nexo Y409V A485L supplied by New England Biolabs). This solution is thenadded to the beads and mixed thoroughly and incubated at 65° C. taking 5μL samples at 3 and 10 minutes and stopping with 5 μL of gel loadingbuffer (xylene cyanol-bromophenol blue dye solution, Sigma-Aldrich). Thereaction mixture is removed from the remaining beads and the beadswashed 3 times with 100 μL of TE buffer.

Chase Step

A sample was removed from the incorporation reaction and added to 1 μMof dNTPs (0.25 μM each). This was stopped after 10 minutes by adding 5μL of gel loading buffer.

Deblocking Step

50 μL of Tris-(2-carboxyethyl)phosphine, trisodium salt (TCEP) 0.1M isadded to the beads and mixed thoroughly. The mixture was then incubatedat 65° C. for 15 minutes. The deblocking solution is removed and thebeads washed 3 times with 100 μL TE buffer. The beads were thenresuspended in 50 μL of TE and a 5 μL sample was removed and 5 μL of gelloading buffer)

Incorporation of the 2^(nd) FFN

20 μL reaction, Tris.HCl pH 8.8 50 mM, Tween-20, 0.01%, MgSO₄ 4 mM,MnCl₂, 0.4 mM, add 2 μM FFN and 100 nM polymerase (Thermococcus sp. 99Nexo Y409V A485L supplied by New England Biolabs). This solution is thenadded to the beads and mixed thoroughly and incubated at 65° C. taking 5μL samples at 3 and 10 minutes and stopping with 5 μL of gel loadingbuffer.

Chase Step

A sample was removed from the incorporation reaction and added to 1 μMof dNTPs (0.2 μM each). This was stopped after 10 minutes by adding 5 μLof gel loading buffer.

Before loading the samples onto a denaturing 12% acrylamide sequencinggel 0.5 μL of EDTA (0.5 M) was added to each sample and then heated to95° C. for 10 minutes.

All patents, patent applications, and published references cited hereinare hereby incorporated by reference in their entirety. While thisinvention has been particularly shown and described with references topreferred embodiments, it will be understood by those skilled in the artthat various changes in form and details may be made without departingfrom the scope of the invention encompassed by the claims.

1. (canceled)
 2. (canceled)
 3. An oligonucleotide comprising anucleotide residue, wherein said nucleotide residue comprises thestructure:

wherein Z is —CH₂N₃; B is a nucleobase selected from the groupconsisting of a purine, a pyrimidine and a deazapurine; and Linker-Labelcomprises

wherein R is an optionally present electron-withdrawing group; Fluor isa fluorophore; and the dotted line connecting the Fluor and the benzenering indicates that substitution may be at any free position on thebenzene ring and further atoms not shown may be present.
 4. Theoligonucleotide of claim 3, wherein R is not present.
 5. Theoligonucleotide of claim 4, wherein Linker-Label comprises


6. The oligonucleotide of claim 5, wherein the dotted line connectingthe Fluor and the benzene ring comprises one or more spacer units suchthat the Fluor is held a sufficient distance from the nucleobase so asnot to interfere with any interaction between the nucleotide and anenzyme.
 7. The oligonucleotide of claim 6, wherein the spacer unitcomprises —O—, —NH—, or —N(alkyl)-.
 8. The oligonucleotide of claim 5,wherein the Linker-Label comprises:

wherein the double asterisks ** indicates the attachment point to thenucleobase.
 9. The oligonucleotide of claim 8, wherein the Fluorcomprises a cyanine, a rhodamine, or a coumarin dye.
 10. Theoligonucleotide of claim 9, wherein the Fluor is a rhodamine dye. 11.The oligonucleotide of claim 9, wherein the Fluor is a Cy5 dye.
 12. Theoligonucleotide of claim 9, wherein the Fluor is attached to the Linkerby reacting an N-hydroxysuccinimide ester of the Fluor with an aminomoiety of the Linker.
 13. The oligonucleotide of claim 8, wherein thenucleobase is

wherein the asterisk * indicates the attachment point to the sugarmoiety of the nucleotide residue.
 14. The oligonucleotide of claim 13,wherein the nucleobase is

and the Fluor is a Cy5 dye.
 15. The oligonucleotide of claim 13, whereinthe nucleobase is

and the Fluor is a rhodamine dye.
 16. The oligonucleotide of claim 8,wherein the nucleotide residue comprises the structure:


17. The oligonucleotide of claim 8, wherein the oligonucleotide is incontact with an aqueous solution comprising ethylenediaminetetraaceticacid (EDTA).
 18. The oligonucleotide of claim 8, wherein theoligonucleotide is in contact with a mutant polymerase.
 19. Theoligonucleotide of claim 8, wherein the oligonucleotide is in contactwith a water soluble phosphine.
 20. The oligonucleotide of claim 8,wherein the oligonucleotide is hybridized to a target polynucleotide,and wherein the target polynucleotide is immobilized to a solid support.21. The oligonucleotide of claim 20, wherein the solid support comprisesa plurality of immobilized target polynucleotide, wherein at least oneimmobilized target polynucleotide is hybridized to a secondoligonucleotide, and wherein the second oligonucleotide comprises adifferent fluorophore than the Fluor.
 22. The oligonucleotide of claim21, the solid support comprises a planar surface.